HIGHLY SENSITIVE AND SPECIFIC DETECTION OF P-GLYCOPROTEIN FUNCTION FOR HEMATOLOGICAL AND SOLID TUMOR-CELLS USING A NOVEL NUCLEIC-ACID STAIN

Progress in our understanding of the contribution of P-glycoprotein (P -gp)-mediated resistance to chemotherapy failure in haematological as well as solid tumours has been hampered by the lack of highly sensitiv e, reliable methods for the detection of P-gp function in fresh human tumour cells. The present study identifies the novel nucleic acid stai n SYTO16 as a highly sensitive and specific dye to assess P-gp functio n. The effect of P-gp is expressed here as the ratio of dye fluorescen ce (RF) from cells incubated with dye with or without 2 mu M of the P- gp inhibitor PSC 833. Using flow cytometric analysis, an RF of 0.9 was found for SYTO16 in the KB3-1 (P-gp(-)) and 1.6 in KB8 (P-gp(+)) cell s. Three types of patients' cells were studied: (1) in haematopoietic CD34(+) cells, which are known to express P-gp, the RF was 6.0 for SYT O16 compared with 2.5 for rhodamine 123 and 1.3 for daunorubicin (mean of five individuals); (2) in acute myeloid leukaemia cells, the RF fo r SYTO16 was 1.0 in P-gp-and 4.5 in P-gp(+) samples; (3) for the first time, we have quantitated P-gp function in fresh human solid tumour ( sarcoma) cells. We found, in a P-gp(+) leiomyosarcoma, an RF of 16 for SYTO16 and 2.7 for daunorubicin. This means that complete inhibition of P-gp function in these sarcoma cells would lead to an increase of d aunorubicin accumulation with 170% compared with 30% in the CD34(+) ce lls. Next, we showed that SYTO16 could be fixed in nuclei by 3.6% form aldehyde treatment, allowing quantification of the nuclear fluorescenc e on cytospins by laser scanning microscopy. In conclusion, SYTO16 pro ved to have a combination of favourable properties: it can be excited at 488 nm and has large fluorescence enhancement upon binding to nucle ic acids, allowing the use of low, nontoxic (< 10 nM) concentrations. Because the RF for SYTO16 is much higher than for daunorubicin, it can be applied for the determination of P-gp function in relatively small numbers of low-P-gp-expressing tumour cells by laser scanning microsc opy. Individual sarcomas were found to have high P-gp function compare d with CD34(+) cells. This assay may be used to select patients for P- gp modulation protocols.